Methylen-oxindole derivatives compositions and tyrosine kinase inhibition therewith

ABSTRACT

PCT No. PCT/EP92/01569 Sec. 371 Date Mar. 12, 1993 Sec. 102(e) Date Mar. 12, 1993 PCT Filed Jul. 10, 1992 PCT Pub. No. WO93/01182 PCT Pub. Date Jan. 21, 1993.The invention provides new methylen-indole derivatives of formula (I)    &lt;IMAGE&gt;  (I)  wherein R is a group   &lt;IMAGE&gt;    in which R4 is hydrogen, hydroxy, C1-C6 alkoxy, C2-C6 alkanoyloxy, carboxy, nitro or NHR7, wherein R7 is hydrogen or C1-C6 alkyl; R5 is hydrogen, C1-C6 alkyl or halogen; and R6 is hydrogen or C1-C6 alkyl; n is zero, 1 or 2; R1 is hydrogen, C1-C6 alkyl or C2-C6 alkanoyl; R2 is hydrogen, C1-C6 alkyl, halogen, cyano, carboxyl, nitro or -NHR7 in which R7 is as defined above; R3 is hydrogen, C1-C6 alkyl or C2-C6 alkanoyl; and the pharmaceutically acceptable salts thereof; and wherein, when, at the same time, R2 is hydrogen, C1-C6 alkyl, halogen or cyano and R3 is hydrogen, R1 and n being as defined above, then at least one of R4, R5 and R6 is other than hydrogen, which are useful as tyrosine kinase inhibitors.

The present invention relates to new 3-methylene-2-oxindole derivatives,to a process for their preparation, to pharmaceutical compositionscontaining them and to their use as therapeutic agents.

The present invention provides compounds having the following generalformula (I) ##STR3## wherein

R is a group ##STR4## in which R₄ is hydrogen, hydroxy, C₁ -C₆ alkoxy,C₂ -C₆ alkanoyloxy, carboxy, nitro or NHR₇, wherein R₇ is hydrogen or C₁-C₆ alkyl;

R₅ is hydrogen, C₁ -C₆ alkyl or halogen; and

R₆ is hydrogen or C₁ -C₆ alkyl;

n is zero, 1 or 2;

R₁ is hydrogen, C₁ -C₆ alkyl or C₂ -C₆ alkanoyl;

R₂ is hydrogen, C₁ -C₆ alkyl, halogen, cyano, carboxyl, nitro or --NHR₇in which R₇ is as defined above;

R₃ is hydrogen, C₁ -C₆ alkyl or C₂ -C₆ alkanoyl; and thepharmaceutically acceptable salts thereof; and wherein, when, at thesame time, R₂ is hydrogen, C₁ -C₆ alkyl, halogen or cyano and R₃ ishydrogen, R₁ and n being as defined above, then at least one of R₄, R₅and R₆ is other than hydrogen. In the compounds of the present inventioneach of the substituents R, --OR₁ and R₂ may be independently on eitherof the benzene or pyrrole moiety of the condensed indole ring system.

The invention includes within its scope all the possible isomers,stereoisomers, in particular Z and E isomers and their mixtures, and themetabolites and the metabolic precursors or bio-precursors (otherwiseknown as pro-drugs) of the compounds of formula (I).

The substituent R is preferably linked to position 2 or 3 of the indolering, in particular to position 3.

When n is 2, each of the --OR₁ groups may be the same or different.

A substituent --OR₁ is preferably linked to position 4, 5, 6 or 7, inparticular to position 5 or 7.

The substituent R₂ is preferably on the benzene ring moiety, inparticular linked to position 5.

Of course only one of the substituents R, --OR₁ and R₂ can be linked tothe same position in the indole ring system. The substituent R₄ ispreferably linked to position 4 or 5, in particular to position 5.

When R₄ is carboxyl, nitro or --NHR₇, in which R₇ is as defined above,the R₂ substituent preferably has not the same meanings. Vice versa,when R₂ is carboxyl, nitro or --NHR₇, in which R₇ is as defined above,the R₄ substituent preferably is other than carboxy, nitro or --NHR₇.

The alkyl groups, and the alkyl moiety in the alkanoyl groups, may bebranched or straight alkyl chain. A C₁ -C₆ alkyl group is preferably aC₁ -C₄ alkyl group, e.g. methyl, ethyl, propyl, isopropyl, butyl,sec-butyl or tert-butyl, in particular methyl or ethyl. A C₂ -C₆alkanoyl group is preferably a C₂ -C₄ alkanoyl group, in particularacetyl, propionyl or butyryl. A halogen is, preferably, chlorine,bromine or fluorine, in particular bromine.

Pharmaceutically acceptable salts of the compounds of the inventioninclude acid addition salts, with inorganic, e.g. nitric, hydrochloric,hydrobromic, sulphuric, perchloric and phosphoric acids, or organic,e.g. acetic, propionic, glycolic, lactic, oxalic, malonic, malic,maleic, tartaric, citric, benzoic, cinnamic, mandelic and salicylicacids, and salts with inorganic, e.g. alkali metal, especially sodium orpotassium, bases or alkaline-earth metal, especially calcium ormagnesium bases, or with organic bases, e.g. alkylamines, preferablytriethyl-amine.

As stated above the present invention also includes within its scopepharmaceutically acceptable bio-precursors (otherwise known aspro-drugs) of the compounds of formula (I), i.e. compounds which have adifferent formula to formula (I) above but which nevertheless uponadministration to a human being are converted directly or indirectly invivo into a compound of formula (I). Preferred compounds of theinvention are the compounds of formula (I), wherein

R is as defined above, R₄ is hydroxy, amino, nitro or carboxy and R₅ andR₆ are hydrogen;

R₁ is hydrogen or C₁ -C₆ alkyl;

n is zero or 1;

R₂ is hydrogen, carboxy, amino or nitro;

R₃ is hydrogen; and the pharmaceutically acceptable salts thereof.

More preferred compounds of the invention are the compounds of formula(I) in which

R is as defined above, R₄ is hydroxy, amino or carboxy; and R₅ and R₆are hydrogen; n is 0 or 1; R₁ is hydrogen; R₂ is hydrogen, amino orcarboxy; R₃ is hydrogen; and the pharmaceutically acceptable saltsthereof.

Examples of specific compounds of the invention are the followingcompounds which, when appropriate, may be either Z- or E-diastereomersor Z,E-mixtures of said diastereomers:

5-hydroxy-3- [(3'-indolyl)methylene]-2-oxindole;

3-[(5'-carboxy-3'-indolyl )methylene]-2-oxindole;

3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;

5-carboxy-3-[(3'-indolyl)methylene]-2-oxindole;

5-amino-3-[(3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

3-[(5',7'-dihydroxy-3'-indolyl)methylene]-2-oxindole;

5-amino-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;

5-carboxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole;

5-amino-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-carboxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

and, if the case, the pharmaceutically acceptable salts thereof.

The compounds of the invention, and the pharmaceutically acceptablesalts thereof, can be obtained by a process comprising the condensationof an aldehyde of formula (II) ##STR5## wherein R₁, R₂, R₃ and n are asdefined above, with a compound of formula (III) ##STR6## wherein R₄, R₅and R₆ are as defined above; and, if desired, converting a compound offormula (I) into another compound of formula (I), and/or, if desired,converting a compound of formula (I) into a pharmaceutically acceptablesalt thereof, and/or, if desired, converting a salt into a freecompound, and/or, if desired, separating a mixture of isomers of acompound of formula (I) into the single isomers. Each of thesubstituents R₂, --OR₁ and --CHO in a compound of formula (II) may beindependently on either of the benzene or pyrrole moiety of the indolering.

The reaction of a compound of formula (II) with a compound of formula(III) is an analogy process which can be carried out according to knownmethods, as herebelow described; preferably in the presence of a basiccatalyst, e.g. pyridine, piperidine, dimethylamine, or a suitable alkalimetal hydroxide or alkoxide.

For example the reaction of a compound of formula (II) with a compoundof formula (III) may be carried out under the conditions of theKnoevenagel reactions as described e.g. by G. Jones in Organic Reactions15, 204 (1967). Suitable catalyst are organic bases such as pyridine,piperidine or diethylamine. The condensation may be performed in aninert organic solvent e.g. pyridine, ethanol, methanol, benzene ordioxane at temperatures ranging from about 0° C. to about 100° C.

Preferably the reaction is carried out in warm ethanol solution in thepresence of piperidine catalyst.

A compound of formula (I) can be converted into another compound offormula (I) according to known methods. For example thede-etherification of a compound of formula (I), wherein one or more R₁substituents are C₁ -C₆ alkyl, so as to obtain a compound of formula (I)wherein one or more R₁ substituents are hydrogen may be performed bywell known methods in organic chemistry. In the case of a phenolicmethyl ether the cleavage can be carried out for example with borontribromide as described by J. F. N. McOmie in Tetrahedron 24, 2289(1968). It is advisable to use about 1 mole of boron tribromide for eachether group together with an extra mol of reagent for each groupcontaining a potentially basic nitrogen or oxygen. The reaction may beperformed in an inert organic solvent such as methylene chloride,pentane or benzene under an inert, e.g. nitrogen, atmosphere attemperatures ranging from about -78° C. to about room temperature.

The acylation of a compound of formula (I) wherein one or more of --OR₁and/or R₄ is hydroxy, so as to obtain a corresponding compound offormula (I) wherein one or more of --OR₁ and/or R₄ is a C₂ -C₆alkanoyloxy group, may be obtained by reaction with a reactivederivative of a suitable carboxylic acid, such as an anhydride orhalide, in the presence of a basic agent, at temperatures ranging fromabout 0° C. to about 50° C. Preferably the acylation is carried out byreaction with the respective anhydride in the presence of an organicbase, such as pyridine.

The optional salification of a compound of formula (I) as well as theconversion of a salt into the free compound and the separation of amixture of isomers into the single isomers may be carried out byconventional methods.

For example the separation of a mixture of geometric isomers, e.g. cis-and trans-isomers, may be carried out by fractional crystallization froma suitable solvent or by chromatography, either column chromatography orhigh pressure liquid chromatography.

The compounds of formula (II) may be obtained according to known methodsfrom compounds of formula (IV) ##STR7##

For example the 3-formylindole derivatives of formula (II) can beobtained from a compound (IV) by formylation with N-methylformanilideand phosphorous oxychloride according to the well known Vilsmeyer-Haackmethod (for a review see W. G. Jackson et al., J. Am. Chem. Soc. 1981,103, 533). The 2-formylindole derivatives are obtained when the3-position is occupied.

The compounds of formula (III) and (IV) are known or may be obtained byknown methods from known compounds.

PHARMACOLOGY

The compounds of the invention possess specific tyrosine kinaseinhibiting activity. Hence they can be useful in the treatment of cancerand other pathological proliferative conditions, such as to inhibit thedevelopment of the atheromatous plaque, in mammals, including humans.

Typical therapeutical indications according to the latter use arereocclusion following coronary angioplasty and in general decrease ofcoronary artery disease.

Recent studies on the molecular basis of neoplastic transformation haveidentified a family of genes, designed oncogenes, whose aberrantexpression causes tumorigenesis.

For example, the RNA tumor viruses possess such an oncogene sequencewhose expression determines neoplastic conversion of infected cells.Several of their oncogene-encoded proteins, such as pp60^(v-src),p70^(gag-yes), p130^(gag-fps) and P70^(gag-fgr) display protein tyrosinekinase activity, that is they catalyse the transfer of the α-phosphatefrom adenosine triphosphate (ATP) to tyrosine residues in proteinsubstrate. In normal cells, several growth factor receptors, for examplethe receptors for PDGF, EGF, α-TGF and insulin, display tyrosine kinaseactivity.

Binding of the growth factor (GF) activates the receptor tyrosine kinaseto undergo autophosphorylation and to phosphorylate closely adjacentmolecules on tyrosine. Therefore, it is thought that the phosphorylationof these tyrosine kinase receptors plays an important role in signaltransduction and that the principal function of tyrosine kinase activityin normal cells is to regulate cell growth. Perturbation of thisactivity by oncogenic tyrosine kinases that are either overproducedand/or display altered substrate specificity may cause loss of growthcontrol and/or neoplastic transformation. Accordingly, a specificinhibitor of tyrosine kinases can be useful in investigating themechanism of carcinogenesis, cell proliferation and differentiations andit can be effective in prevention and chemotherapy of cancer and inother pathological proliferative conditions.

The tyrosine specific protein kinase activity of these compounds isshown by the in vitro tests described herebelow.

v-abl kinase purification

The enzyme used in the Abelson tyrosine kinase p95^(v-ab) which wasproduced and isolated as follows: 1 Liter cultures of HB-130 cells in LBmedium, supplemented with ampicillin, were grown at 30° C. as describedby Wang et al. in J. Biol. Chem. 260, 64 (1985). The expression of v-ablprotein was induced by shifting the temperature to 42° C. for 3-4 h. Thebacterial cells were collected on ice, centrifuged and frozen in liquidnitrogen. The cell pellet was lysed as described by Ferguson et al. inJ. Biol. Chem. 260, 3652 (1985) and in Biochem. J. 257, 321 (1989).Briefly, bacterial proteins were removed by differential solubilizationwith sodium deoxycholate, NaCl and β-octylglucopyranoside in differentsteps. Insoluble v-abl protein was solubilized with 2M KSCN and dialyzedagainst 100 volumes of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA and 0.1 mMdithiothreitol (buffer A). The soluble proteins were separated bychromatography on a prepacked Mono-Q anion exchange column in a f.p.l.c.system. The v-abl activity was eluted with a linear KCl gradient (O-I Mbuffer A). Active fractions were pooled, made 50% in glycerol and storedin small aliquots at -20° C.

Myelin Basic Proteins phophorylation assay

The in vitro test with Myelin Basic Protein as substrate was carried outas follows: Protein phosphorylation was performed by incubating 40 ng ofpurified v-abl kinase, 1.5 μCi [α-³² p] ATP, 10 μM cold ATP, 56 μMmyelin basic protein in 50 μl of Tris-HCl 25 mM, pH 8.0, containingMgCl₂ 10 mM, dithiothreitol 0.1 mM (kinase buffer) at 22° C. Thereaction was stopped by addition of equal volumes of 2-fold concentratedLaemmli electrophoresis buffer [U. K. Laemmli, Nature (London) 230, 680(1970). Samples were boiled again for 3 min and separated in SDS-PAGE(15% acrylamide). Gels were dried and exposed to autoradiographic filmsfor 15-30 min at -70° C. Bands were located by autoradiography, excisedfrom gel and counted in a liquid scintillation counter.

Autophosphorylation assay

For the autophosphorylation assay the v-abl kinase wasimmunoprecipitated with antiphosphotyrosine antibodies and the resultingimmunocomplex analyzed in 50 μl of kinase buffer in the presence of 10μM ATP and 10 μCi [α-³² P]-ATP. The reaction was stopped after 15 min atroom temperature with boiling Laemmli buffer. Samples were boiled againfor 3 min and separated in SDS-PAGE 18% acrylamide). Gels were dried andexposed to autoradiographic films for up to 3 h at -70° C. Bands werelocated by autoradiography, excised and counted as above. In table I andII a representative compound of this invention is compared with thecorresponding non hydroxylated analog which is encompassed by thegeneral formula of patent application WO91/13055. The comparison showsthat the introduction of an hydroxyl group, while increasing onlyslightly the potency toward exogenous substrates significantly enhancesthe inhibitory activity on the autophosphorylation.

                  TABLE I                                                         ______________________________________                                        Myelin Basic Protein phosphorylation assay.                                                           IC.sub.50 (μM)                                     ______________________________________                                        5-hydroxy-3-[(3'-indolyl)methylen]-2-oxindole                                                           0.4                                                 3-[(3'-indolyl)methylen]-2-oxindole                                                                     0.6                                                 ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Autophosphorylation assay.                                                                            IC.sub.50 (μM)                                     ______________________________________                                        5-hydroxy-3-[(3'-indolyl)methylen]-2-oxindole                                                           0.4                                                 3-[(3'-indolyl)methylen]-2-oxindole                                                                     10                                                  ______________________________________                                    

In view of their high activity and low toxicity, the compounds of theinvention can be used safely in medicine. For example, the approximateacute toxicity (LD₅₀) of the compounds of the invention in the mouse,determined by single administration of increasing doses and measured onthe seventh day after the treatment was found to be negligible.

The compounds of the invention can be administered in a variety ofdosage forms, e.g. orally, in the form of tablets, capsules, sugar orfilm-coated tablets, liquid solutions or suspensions; rectally, in theform of suppositories; parenterally, e.g. intramuscularly, or byintravenous injection or infusion; or topically. The dosage depends onthe age, weight, conditions of the patient and administration route; forexample, the dosage adopted for oral administration to adult humans mayrange from about 10 to about 150-200 mg per dose, from 1 to 5 timesdaily. Of course, these dosage regimens may be adjusted to provide theoptimal therapeutic response.

The invention includes pharmaceutical compositions comprising a compoundof formula (I) or a pharmaceutically acceptable salt thereof inassociation with a pharmaceutically acceptable excipient (which can be acarrier or diluent).

Object of the present invention is also the use of a compound of formula(I), as defined above, or a pharmaceutically acceptable salt thereof,for the manufacture of a medicament for use as tyrosine kinaseinhibitor, in particular as anti-cancer and anti-proliferative agent.The pharmaceutical compositions containing the compounds of theinvention are usually prepared following conventional methods and areadministered in a pharmaceutically suitable form.

For example, the solid oral forms may contain, together with the activecompound, diluents, e.g., lactose, dextrose, saccharose, cellulose, cornstarch or potato starch; lubricants, e.g. silica, talc, stearic acid,magnesium or calcium stearate, and/or polyethylene glycols; bindingagents, e.g. starches, arabic gums, gelatin, methylcellulose,carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents,e.g. a starch, alginic acid, alginates or sodium starch glycolate,effervescing mixtures; dyestuffs; sweeteners; wetting agents, such aslecithin, polysorbates, laurylsulphates; and, in general, non-toxic andpharmacologically inactive substances used in pharmaceuticalformulations. Said pharmaceutical preparations may be manufactured inknown manner, for example, by means of mixing, granulating, tabletting,sugar-coating or film-coating processes.

The liquid dispersion for oral administration may be, e.g., syrups,emulsions and suspensions.

The syrup may contain as carrier, for example, saccharose or saccharosewith glycerine and/or mannitol and/or sorbitol.

The suspensions and the emulsions may contain as carrier, for example, anatural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose or polyvinyl alcohol.

The suspensions or solutions for intramuscular injections may contain,together with the active compound, a pharmaceutically acceptablecarrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g.propylene glycol, and, if desired, a suitable amount of lidocainehydrochloride.

The solutions for intravenous injections or infusion may contain ascarrier, for example, sterile water or, preferably, they may be in theform of sterile, aqueous, isotonic saline solutions.

The suppositories may contain, together with the active compound, apharmaceutically acceptable carrier, e.g. cocoa-butter, polyethyleneglycol, a polyoxyethylene sorbitan fatty acid ester surfactant orlecithin. Compositions for topical application, e.g. creams, lotions, orpastes, can be prepared by admixing the active ingredient with aconventional oleaginous or emulsifying excipient.

A further object of the present invention is a combined method oftreatment of cancer in mammals, including humans, in need of suchtreatment, said method comprising administering

1) a compound of formula (I), or a pharmaceutically acceptable saltthereof, and

2) an additional antitumor agent, in amounts and close enough togetherin time sufficient to produce a therapeutically useful effect.

Object of the present invention is also to provide products containing acompound of formula (I) , or a pharmaceutically acceptable salt, and anadditional antitumor agent as a combined preparation for simultaneous,separate or sequential use in anti-cancer therapy.

The term "antitumor agent" is meant to comprise both a single antitumordrug and "cocktails" i.e. a mixture of such drugs, according to theclinical practice.

Antitumor agents that can be formulated with a compound of the inventionor, alternatively, can be administered in a combined method oftreatment, are e.g. doxorubicin, daunomycin, epirubicin, idarubicin,etoposide, fluorouracil, mephalan, cyclophosphamide, bleomycin,vinblastin and mitomycin or a mixture of two or more thereof.

The compounds of the invention can therefore be used in a treatment toameliorate a cancer. They may be administered to a patient sufferingfrom a cancer treatable with an antitumor agent, for example ananthracycline glycoside such as doxorubicin, daunomycin, epirubicin oridarubicin as mentioned above, together with the antiproliferativeagent. A compound of the invention and an antitumor agent such as ananthracycline glycoside can be administered to improve the condition ofa patient having a leukaemia such as myeloblastic leukaemia, lymphoma,sarcoma, neuroblastoma, Wilm's tumor or malignant neoplasm of thebladder, breast, lung or thyroid.

The following examples illustrate but do not limit the invention.

EXAMPLE 1 5-Hydroxy-3-[(3'-indolyl)methylene]-2-oxindole [I, R asdefined, n=0, R₂ =R₃ =R₅ =R₆ =H, R₄ =5-OH]

A solution of 3-indolecarboxaldehyde (145 mg, 1 mmol),5-hydroxy-2-oxindole (149 mg, 1 mmol) and piperidine (60 mg, 0.7 mmol)in absolute ethanol (10 ml) is heated for 3 h at 60° C. under nitrogen.Then the reaction mixture is chilled and evaporated under vacuum todryness. The residue is submitted to column chromatography over silicagel using methylene-chloride/ethanol 4% as eluant. Pure title compoundis so obtained in 60% yield (166 mg). Alternatively, the reactionmixture is concentrated under vacuum and then chilled to 0°-5° C., theprecipitate filtered, the residue washed with ice-cooled ethanol andfinally dried under vacuum. Compounds of higher purity are obtained byfurther crystallization from ethanol.

C₁₇ H₁₂ N₂ O₂ requires: C 73.89 H 4.38 N 10.14 found: C 73.51 H 4.21 N9.92 MS m/z: 276 IR cm⁻¹ (KBr): 3600-2500 (NH, OH), 1650 (CO), 1600,1580, 1530, 1480 (C═C) m.p. 293° C. (dec.).

According to the above described procedure, the following compounds canbe prepared:

5-carboxy-3-[(3'-indolyl)methylene]-2-oxindole;

5-amino-3- [3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3- [(5'hydroxy-3'-indolyl)methylene]-2-oxindole;

MS m/z: 282 IR cm⁻¹ (KBr): 3600-2600 (NH, OH), 1655 (CO), 1605, 1585,1535 (C═C)

5-hydroxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

3-[(5',7'-dihydroxy-3'-indolyl)methylene]-2-oxindole;

5-amino-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;

5-carboxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-hydroxy-3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole;

5-amino-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-carboxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;

5-methoxy-3-[(5'-methoxy-3'-indolyl)-methylene]-2-oxindole;

5-acetoxy-3-[(5'-acetoxy-3'-indolyl)methylene]-2-oxindole;

3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole,

C₁₈ H₁₂ N₂ O₃ requires: C 71.04; H 3.98; N 9.21 found: C 70.83; H 4.6; N8.85 MS m/z: 304; m.p.>330° dec. IR cm⁻¹ (KBr): 3600-3000 (NH),3000-2100 (OH), 1710 (CO), 1640, 1620, 1600, 1550 (arom);

3-[(5'-amino-3'-indolyl)methylene]-2-oxindole,

C₁₇ H₁₃ N₃ O requires: C 74.14; H 4.71; N 15.26 found: C 73.88; H 4.51;N 14.91 MS m/z 275; m.p. 250° dec. IR cm⁻¹ (KBr) : 3300, 2380 (NH), 1670(CO), 1600, 1510 (C═C);

3-[(5'-nitro-3'-indolyl)methylen]-2-oxindole,

C₁₇ H₁₁ N₃ O₃ requires: C 66.88; H 3.63; N 13.76 found: C 66.58; H 3.74;N 13.64 MS m/z 305; m.p.>350° C. IR cm⁻¹ (KBR): 3350, 3230 (NH), 1680(CO), 1620, 1605, 1580 (C═C), 1530, 1340 (NO₂);

3- [(1'-methyl-3'-indolyl)methylen]-2-oxindole,

C₁₈ H₁₄ N₂ O requires: C 78.81; H 5.14; N 10.21 found: C 78.42; H 5.17;N 10.00 MS m/z 274 m.p. 230° C. (dec.) IR cm⁻¹ (KBr): 3300-2000 (NH),1680 (CO), 1610, 1600, 1570, 1500 (C═C);

3-[(3'-indolyl)methylen]-1-methyl-2-oxindole,

C₁₈ H₁₄ N₂ O requires: C 78.81; H 5.14; N 10.21 found: C 78.61; H 5.16;N 10.23 MS m/z 274 m.p. 274° C. IR cm⁻¹ (KBr): 3220 (NH), 1675 (CO),1605, 1500, 1490 (C═C).

EXAMPLE 2 5-Hydroxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole [I,R as defined, n=1, R₁ =R₂ =R₃ =R₅ =R₆ =H, R₄ =5-OH]

The starting material for this de-etherification example is5-methoxy-3-[(5'-methoxy-3'-indolyl)methylene]-2-oxindole, which can beobtained according to the procedure described in Example 1.

To a stirred solution of 5-methoxy-3-[(5'-methoxy-3'-indolyl)methylene]-2-oxindole (310 mg, 1 mmol) in anhydrous dichloromethane (10ml) is added at -78° C. under nitrogen, over a period of 10 min, a 1.0Msolution of boron tribromide in dichloromethane (3 ml, 3 mmol). Theresulting mixture is stirred for another 1 h at -78° C. and then allowedto warm to room temperature. After stirring for 1.5 h at 20°-25° C. themixture is cooled to -10° C. and then quenched by the dropwise additionof water (10 ml) over a 10-min period. After addition of ethylacetatethe organic layer is separated, washed with water, dried with Na₂ SO₄and evaporated under vacuum to dryness. The residue is crystallized fromethanol thus giving 198 mg of pure title compound (yield 70%).

C₁₇ H₁₂ N₂ O₃ requires: C 72.33 H 4.29 N 6.38 found: C 72.11 H 4.07 N6.29 MS m/z: 282 IR cm⁻¹ (KBr): 3600-2600 (NH, OH), 1655 (CO), 1605,1585, 1535 (C═C)

According to the above described procedure and starting from thecorresponding methylether, the hydroxyl compounds mentioned in Example 1can be obtained.

EXAMPLE 3 5-Acetoxy-3-[(5'-acetoxy-3'-indolyl)methylene]-2-oxindole [I,R as defined, n=1, R₁ =Ac, R₄ =5-OAc, R₂ =R₃ =R₅ =R₆ =H]

The starting material for this acylation example is5-hydroxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole, which may beobtained according to the procedure described in Examples 1 and 2.

To a cooled solution of 5-hydroxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole (282 mg, 1 mmol) in dry pyridine (0.5 ml) is addedacetic anhydride (306 mg, 3 mmol) and the mixture maintained at 0°-5° C.overnight. Thereupon the mixture concentrated under vacuum, the residuedissolved in dichloromethane, the organic layer washed with water andthen evaporated under reduced pressure. The crude product iscrystallized from chloroform/methanol to yield pure title compound in80% yield (301 mg).

C₂₁ H₁₆ N₂ O₅ requires: C 67.02 H 4.29 N 7.44 found: C 66.91 H 4.05 N7.29 MS m/z: 376 IR cm⁻¹ (KBr): 3600-3200 (NH), 1750 (CH₃ COO), 1650(CONH), 1600, 1580, 1530 (C═C)

According to the above described procedure, the hydroxyl compoundsobtained in Example 1 and 2 can be transformed into the corresponding C₂-C₆ alkanoyloxy derivatives.

EXAMPLE 4 5-Nitro-3-indolealdehyde. [II, n=0, R₂ =5-NO₂, R₃ =H]

A mixture of N-methylformanilide (176 mg, 1.3 mmol) and phosphorousoxychloride (199 mg, 1.3 mmol) is stirred for 15 min at 20°-25° C. undernitrogen. then a solution of 5-nitroindole (162 mg, 1 mmol) in1,2-dichloroethane (5 ml) is added and the mixture heated to reflux for3 h. After cooling the mixture is poured onto iced water, theprecipitate filtered off and washed with water. Thereupon the residue ischromatographed over silica gel using benzene/ethyl acetate as eluant.Thus pure title compound is obtained in 80% yield (152 mg).

C₉ H₆ N₂ O₃ requires: C 56.85 H 3.18 N 14.73 found: C 56.79 H 3.01 N14.51 MS m/z: 190 IR cm⁻¹ (KBr): 3140, 3090 (NH), 1650 (CO), 1511, 1345(NO₂)

These nitro-intermediates besides leading to final products of formula(I) with R₂ =NO₂ give also rise to final products with R₂ =NH₂obtainable from the former by reduction. By proceding analogously, thefollowing protected intermediates can be prepared, which afterdeprotection at a suitable stage of the synthesis give also rise tofinal product of formula (I) with free carboxyl (R₂ =COOH) and freehydroxyl (R₂ =OH) respectively:

3-carbomethoxy-3-indolealdehyde; and

3-methoxy-3-indolealdehyde.

EXAMPLE 5

Tablets each weighing 0.150 g and containing 25 mg of the activesubstance, can be manufactured as follows: composition (for 10,000tablets):

    ______________________________________                                        5-Hydroxy-3-[(3'-indolyl)methylene]-                                                                 250 g                                                  2-oxindole                                                                    Lactose                800 g                                                  Corn starch            415 g                                                  Talc powder             30 g                                                  Magnesium stearate      5 g                                                   ______________________________________                                    

The 5-hydroxy-3-[(3'-indolyl)methylene]-2-oxindole, the lactose and halfthe corn starch are mixed; the mixture is then forced through a sieve of0.5 mm mesh size.

Corn starch (10 g) is suspended in warm water (90 ml) and the resultingpaste is used to granulate the powder. The granulate is dried,comminuted on a sieve of 1.4 mm mesh size, then the remaining quantityof starch, talc and magnesium stearate are added, carefully mixed andprocessed into tablets.

EXAMPLE 6

Capsules, each dosed at 0.200 g and containing 20 mg of the activesubstance can be prepared.

Composition for 500 capsules:

    ______________________________________                                        3-[(5'-amino-3'-indolyl)methylene]-                                                                  10 g                                                   2-oxindole                                                                    Lactose                80 g                                                   Corn starch             5 g                                                   Magnesium stearate      5 g                                                   ______________________________________                                    

This formulation is encapsulated in two-piece hard gelatin capsules anddosed at 0.200 g for each capsule.

We claim:
 1. A compound of formula (I): ##STR8## in which R₄ ishydrogen, hydroxy, C₁ -C₆ alkoxy, C₂ -C₆ alkanoyloxy, carboxy, nitro orNHR₇, wherein R₇ is hydrogen or C₁ -C₆ alkyl;R₅ is hydrogen, C₁ -C₆alkyl or halogen; and R₆ is hydrogen or C₁ -C₆ alkyl; n is zero, 1 or 2;R₁ is hydrogen, C₁ -C₆ alkyl or C₂ -C₆ alkanoyl; R₂ is hydrogen, C₁ -C₆alkyl, halogen, cyano, carboxyl, nitro or --NHR₇ in which R₇ is asdefined above; R₃ is hydrogen, C₁ -C₆ alkyl or C₂ -C₆ alkanoyl; or apharmaceutically acceptable salt thereof; and wherein: (i) when, at thesame time, R₂ is hydrogen, C₁ -C₆ alkyl, halogen or cyano and R₃ ishydrogen, R₁ and n being as defined above, then at least one of R₄, R₅and R₆ is other than hydrogen; and (ii) when n is zero and R₃ and R₆ areeach hydrogen or CH₃, then at least one of R₂, R₄ and R₅ is other thanhydrogen.
 2. A compound of formula (I), according to claim 1, wherein R₄is hydroxy, amino, nitro or carboxy and R₅ and R₆ are hydrogen;R₁ ishydrogen or C₁ -C₆ alkyl; n is zero or 1; R₂ is hydrogen, carboxy, aminoor nitro; R₃ is hydrogen; and the pharmaceutically acceptable saltsthereof.
 3. A compound of formula (I), according to claim 1, wherein R₄is hydroxy, amino or carboxy; and R₅ and R₆ are hydrogen; n is 0 or 1;R₁ is hydrogen; R₂ is hydrogen, amino or carboxy; R₃ is hydrogen; andthe pharmaceutically acceptable salts thereof.
 4. A compound selectedfrom the group consisting of the following which, when appropriate, maybe either Z- or E-diastereoisomers or Z,E-mixtures of saiddiastereoisomers:5-hydroxy-3-[(3'-indolyl)methylene]-2-oxindole;3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole;3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;5-carboxy-3-[(3'-indolyl)methylene]-2-oxindole;5-amino-3-[(3'-indolyl)methylene]-2-oxindole;5-hydroxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-hydroxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-amino-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-hydroxy-3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;5-carboxy-3-[(5'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-hydroxy-3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole;5-amino-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-carboxy-3-[(7'-hydroxy-3'-indolyl)methylene]-2-oxindole;5-methoxy-3-[(5'-methoxy-3'-indolyl)-methylene]-2-oxindole;5-acetoxy-3-[(5'-acetoxy-3'-indolyl)methylene]-2-oxindole;3-[(5'-carboxy-3'-indolyl)methylene]-2-oxindole;3-[(5'-amino-3'-indolyl)methylene]-2-oxindole;3-[(5'-nitro-3'-indolyl)methylene]-2-oxindole;and, if the case, thepharmaceutically acceptable salts thereof.
 5. A pharmaceuticalcomposition comprising a suitable carrier and/or diluent and, as anactive principle, a compound of formula (I) according to claim 1 or apharmaceutically acceptable salt thereof.
 6. A pharmaceuticalcomposition comprising a suitable carrier and/or diluent and, as anactive principle, a compound selected from3-[(1'-methyl-3'-indolyl)methylen]-2-oxindole,3-[(3'-indolyl)methylen]-1-methyl-2-oxindole and the pharmaceuticallyacceptable salts thereof, the compound or salt being either the Z- orE-diastereoisomer or a Z/E mixture of diastereoisomers.
 7. A method oftreating a patient in need of treatment with a tyrosine kinaseinhibitor, the method comprising administering thereto a therapeuticallyeffective amount of a compound selected from the compounds of formula(I) and salts thereof, as claimed in claim 1, and the compounds andsalts defined in claim 6.